Plasminogen assay system

ABSTRACT

PLASMINOGEN IN BLOOD PLASMA IS QUANTITATIVELY DETERMINED BY MEASURING THE DIAMETER OF THE CLEAR RADIAL DIFFUSION ZONE PRODUCED BY THE REACTION OF THE BLOOD PLASMA WITH KNOWN AMOUNTS OF CLOTTED FIBRINOGEN AND PLASMINOGEN SUSPENDED IN A STABLE GEL MEDIUM.

United States Patent US. Cl. 195-99 5 Claims ABSTRACT on THE DISCLOSUREPlasminogen in blood plasma is quantitatively deter mined by measuringthe diameter of the clear radial diffusion zone produced by the reactionof the blood plasma with known amounts of clotted fibrinogen andplasminogen suspended in a stable gel medium.

This is a continuation of application Ser. No. 764,628, filed. Oct. 2,1968, now abandoned.

This invention relates to a method for the determination of fibrinolyticactivity and, more particularly, to a method for the quantitativedetermination of fibrinolytic activity in blood plasma and fractionsthereof.

The clotting of blood is of profound biological significance. Itinvolves a complex mechanism and depends upon the presence and activityof a number of plasma proteins. Of these proteins, fibrinogen is ofprimary importance since the fundamental feature of the clottingmechanism is the conversion of a solution of fibrinogen into a rigidinsoluble fibrin clot. This transformation is normally brought about bythe interaction of fibrinogen and thrombin. Thrombin is derived from itsprecursors, prothrombin. The transformation of .prothrombin intothrombin generally requires the presence of calcium ions and a member ofa group of activating agents known as thromboplastinsg The fibrin clotdisintegrates over a period of time as a result of enzymic action. Theenzyme involved in this acf" tion is referred to as fibrinolysin (orplasmin). This proteolytic enzyme is present in normal blood in the formof its inert precursor, profibrinolysin (or plasminogen). Theplasminogen can be activated or rendered proteolytic by the use ofcertain reagents, for example, chloroform, streptokinase, urokinase, andother such plasminogen activators. These activators cause the conversionof plasminogen into plasmin which attacks the coagulation proteins ofthe fibrin clot. Inhibitors normally present in the blood withplasminogen generally retard this reaction.

The term fibrinolytic system is defined herein as the process ofdisintegration of the fibrin clot and the term fibrinolytic activity isdefined herein to include the above-described chemical and physicalactivities involved in this disintegration.

The known methods for the determination of fibrinolytic activitygenerally employ reaction in a so-called wet system. For example, Astrupet al., Arch. Biochem. and Biophys, vol. 40, pp. 346-351 (1952),disclose a wet fibrin plate system for the determination of plasminactivity in blood.

It is an object of the present invention to provide a new and improvedmethod for the determination of fibrinolytic activity in blood plasmaand fractions thereof.

It is another object of the present invention to provide a novel methodfor the quantitation of the various components of the fibrinolyticsystem.

In accordance with the present invention a stable gel is provided as areaction medium for the fibrinolytic system for the quantitativedetermination of fibrinolytic activity. A fibrinolytic component of thefibrinolytic system comprising clotted fibrinogen is homogeneouslysuspended in Patented Dec. 11, 1973 the gel and allowed to react with anunknown sample comprising blood plasma or a fraction thereof containingother essential components of the fibrinolytic system for apredetermined period of time to produce a clear radial diffusion zone ofreaction product at the interface of the unknown sample and the gel. Thediameter of the radial diffusion zone is directly proportional to thelog of the fibrinolytic activity in the sample being tested. Thefibrinolytic activity in an unknown sample is determined by comparisonwith control samples of known fibrinolytic activity.

The term stable gel is defined herein as a gel that is inert orchemically non-reactive with the components of the fibrinolytic systemsuspended in the gel.

In preparing the gel medium from about 1.0% to about 10.0% by weight ofa gelling agent is dissolved in a heated buffer system and then mixedwith a predetermined amount of a clotted fibrinogen component of thefibinolytic system as hereinbefore stated. Generally, from about 10 mg.percent to about 2 gm. percent, and preferably about mg. percent, offibrinogen is employed in the heated buffer system. While still hot,this mixture is poured into a low-sided flat receptacle (hereinafteralso referred to as a plate) in an amount appropriate for the size ofthe receptacle. The mixture is allowed to gel, and wells or cylindricalholes of approximately 1 to 10 mm. in diameter are punched or otherwiseformed in the gel.

Known and unknown samples, respectively, comprising blood plasma or afraction thereof, containing other essential components of thefibrinolytic system are then added to the open wells in the gel by useof a capillary pipette or similar device and the plate is incubated atabout 20 to 60 C., and preferably at about 37 C., for a predeterminedperiod of time. During this incubation, a fibrinolytic reaction occursas the material in the Wells diifuses outwardly into the gel medium andproduces a clear radial diffusion zone of reaction product.

At the end of the incubation period, the gel is visually inspected,preferably with the assistance of a lighted magnifying viewer having ascale for measurement of the diameter of the radial diffusion zone. 'Bycomparing the diameters of the clear radial diffusion zones producedduring the incubation period by the reaction of the fibrinolyticcomponent in the gel with the fibrinolytic components in known andunknown samples, respectively, the fibrinolytic activity in the unknownsample can be readily determined.

In the preparation of the gel medium, any conventional gelling agent canbe used, for example, gelatin, pectin, silica gel, starch,polysaccharides from seaweed such as agar, algin and carrageenin,synthetic polymeric gelling agents such as the cross-linkedpolyacrylamide disclosed in US. Pat. 3,046,201 and the like materials.The gelling agent preferably has the physical properties characterizingagar-agar insofar as it is readily dispersible in water and capable offorming an essentially clear hydrogel of sufficient rigidity so that thereceptacle or plate containing the gel can be inverted without danger ofthe gel falling out.

A purified agarose is the preferred gelling agent employed in thepresent invention. Agarose is the neutral galactose polymer which hasbeen separated from the agaropectin fraction of agar by any conventionalmethod, for example, the methods described in US. Pats. 3,281,- 409,3,335,127 and 3,362,884. This gelling agent is preferably used at aconcentration of about 2.5% by weight of the gel medium.

The buffer system generally employed in the gel has a pH of from about6.0 to about 8.5 and preferably about 7.3. A preferred buffer comprises0.154 M sodium chloride, 0.04 M imidazole and 0.02 Methylenediaminetetraacetate. Other suitable buffers which can be usedare, for example, tris(hydroxymethyl)aminomethane, phosphate andbarbital buffers.

In order to prepare a plate containing the gel with clotted fibrinogen,suitable clotting can be produced by dipping the plate containing thesolidified gel and fibrinogen in a thrombin solution for an appropriatelength of time, for example, from about 0.1 to about 15 minutes. The gelcontaining the clotted fibrinogen is then ready for punching of thewells as hereinbefore stated.

The gel plate containing the clotted fibrinogen can be overlaid with aprotective membrane, packaged by various means, and thereby madeconveniently available for subsequent use in the determination offibrinolytic activity by hospitals, laboratories and other agencies andpersons having a need for a simplified, yet accurate, determination offibrinolytic activity in blood plasma samples. The packages for theseplates can be, for example, plastic film or metal foil bags, pouches andthe like, preferably sterilized and sealed to prevent the admission ofair, moisture, dirt and other contaminating materials. Suitable metalfoil can be fabricated, for example, from aluminum and the like metals;suitable plastic film can be fabricated, for example, from vinylchloride and vinylidene chloride polymers and copolymers, polyvinylchloride, polyvinyl alcohol, polyethylene, polypropylene, polystyrene,polycarbonates, polyamides, cellulose acetate and propionate, cellulosetriacetate, cellulose acetate butyrate, ethyl cellulose, fluorocarbons,acrylic plastics such as acrylates and methacrylates, and polyesterssuch as, for example, the polyesters formed by condensation reactionsbetween ethylene glycol and terephthalic acid.

The clotted fibrin plates can also be packaged in combination withcontrol samples, buffer materials, capillary tubes and other componentsfor making the complete fibrinolytic assay.

According to two preferred aspects of the present invention, thefibrinolytic component in the gel media and the fibrinolytic activity tobe determined in the unknown sample are as follows:

Plate 1. Cloted fibrinogen is suspended in the gel and determinationsare made for (a) total plasminogen, (b) available plasmin, (c) activeplasmin and (d) fibrinolytic inhibitor in the unknown sample.

Plate II. Clotted fibrinogen and plasminogen are suspended in the geland a determination is made for plasminogen activator in the unknownsample.

In Plate I, a highly purified fibrinogen is used as the fibrinolyticcomponent in the gel..

In Plate II, the fibrinolytic component comprises fibrinogen andplasminogen. This component can be prepared by using a fibrinogen whichis contaminated with a known amount of plasminogen or by using a highlypurified fibrinogen as in Plate I to which a predetermined amount ofplasminogen is added. When a highly purified fibrinogen is employed, itis preferred to add the plasimnogen to a final concentration of fromabout 0.01 to about 20 units/n11. and most preferredly about oneunit/ml. As used herein, one unit/ml. of plasminogen is defined as theamount of plasminogen in pooled normal plasma.

The following examples will further illustrate the invention althoughthe invention is not limited to these specific examples. All parts andpercentages herein are by weight unless otherwise specified.

EXAMPLE 1 An agarose gel plate is prepared as follows:

Preparation of reagents (A) Agarose gel plate buffer-0.154 M sodiumchloride,

0.04 M imidazole and 0.02 M ethylenediaminetetraacetate, pH 7.30. Y

(B) Glycine citrated saline solution-0.123 M sodium chloride, 0.020 Msodium citrate and 0.1 M glycine.

(C) Thrombin solution-Parke-Davis topical thrombin, bovine origin, 5000units/vial, is diluted to 50 units/ 4 ml. with cold distilled water, thepH is adjusted to 5.30 with cold 1.0 N acetic acid, the resultantprecipitate is spun down in a refrigerated centrifuge and the supernateis decanted and lyophilized. The lyophilized product is reconstituted toits original volume with sterile, distilled water prior to use.

Procedure A highly purified fibrinogen solution is prepared as follows:

Pooled human plasma is frozen and then slowly thawed to 4' C. Thecryoprecipitate is removed by centrifugation and dissolved in $4 theoriginal plasma volume of glycine citrated saline. The pH of thesolution is adjusted to 6.50 with 1.0 Normal acetic acid. Then 3.5 gm.percent of polyethylene glycol 4000 (mol. wt. ca. 4000) is slowly addedto the above solution and precipitation is carried out with stirring for15 minutes at room temperature. The resultant precipitate is spun out bycentrifugation and dissolved in glycine citrated saline A the originalplasma volume). The pH of the solution is adjusted to 6.88 with 1.0Normal sodium hydroxide and the solution is then cooled to 9 C. Glycineis slowly added to the solution to make the final glycine concentration1.8 M and the precipitation is carried out with stirring for 45 minutesat 5 C. The resultant precipitate is spun out by centrifugation anddissolved in normal physiologic saline to produce a concentration ofapproximately 2.5 gm. percent fibrinogen. The fibrinogen solution isthen adsorbed three times with 5 gm. percent Darco G-60 charcoal. Eachadsorption is carried out for thirty minutes at room temperature, thesolid charcoal being removed after each process by centrifugation. Thefinal solution is adjusted to 260 mg. percent fibrinogen by appropriatedilution with normal physiologic saline. The solution is then stored inthe frozen state.

An agarose solution is prepared as follows: 2.5 gm. percent agarose iscompletely dissolved in the abovedescribed agarose gel plate bulfer.

A clotted fibrin plate is then prepared as follows:

The hereinbefore prepared agarose solution is brought to 5 35 6 C. andone volume of the solution is homogeneouslymixed with one volume of theabove-described highly purified fibrinogen solution brought to roomtemperature. The mixture is then poured into a plate of approximately 3inches x 1 inch x inch deep and allowed to solidify. The plate with thesolidified gel is then dipped into the above-described thrombin solutionfor an appropriate length of time (about 30 seconds) and removed. Theplate is dipped into a distilled water bath for an appropriate length oftime (about 30 seconds) and then removed. Six wells, each having adiameter of 2 mm. and being equidistantly spaced apart, are then punchedinto the gel in the clotted plate. The plate is then capped with aprotective membrane, packaged and stored at 2 to 8 C.

The clotted fibrin plate is used with plasma or appropriate plasmapreparations for making the following quantitative determinations:

Test I. Total plasminogen is determined in a streptokinase-activatedeuglobulin fraction of plasma.

Test II. Available plasmin is determined in a streptokinase-activatedplasma.

Test III. Active plasmin is determined in plasma.

The procedure employed in carrying out the above three determinations isas follows:

(I) TOTAL PLASMINOGEN TEST (1) Prepare a streptokinase-activatedfraction (SK-E fraction).

(a) Dilute the patients citrated plasma 1:18 with 5 C.

distilled water.

(b) Adjust the pH to 5.3 with 1.0 N acetic acid.

(0) Centrifuge in a refrigerated centrifuge for 20 minutes.

(d) Redissolve the precipitate with sufiicient agarose gel plate bufferto obtain the original plasma volume.

(e) Add 0.1 ml. of streptokinase to 1.0 ml. of the euglobulin fraction(product of Step 1d).

(2) Prepare an activated euglobulin control by adding 1.0 ml. of aeuglobulin control containing a known plasminogen activity to 0.1 ml. ofstreptokinase.

(3) To a total of 6 test tubes, add the following amounts of agarose gelplate buffer:

(4) Incubate the previously prepared SK-E fraction, the activatedeuglobulin control, and the 6 tubes of agarose gel plate buffer at 37 C.for minutes.

(5) Add the following amounts of incubated SK-E fraction and activatedeuglobulin control to the tubes of agarose gel plate buffer and mixwell:

SK-E fraction Activated euglobulin Tube number (ml.) control (ml.)

(6) Open the clotted fibrin plate; remove and discard the protectivemembrane from the agar surface.

(7) Fill three wells of the clotted fibrin plate with the activatedeuglobulin control-agarose gel plate buffer dilutions. Fill theremaining three wells with SK-E fractionagarose gel plate bufferdilutions. Touch the tip of each capillary to the bottom of the well andallow the solution to flow by gravity so that the well is filled to thelevel of the agar surface.

(8) Replace the top cover of the plate and incubate at 37 C., preferablyin a moist chamber, for approximately 2 /2 hours, or until clearreaction zones appear for all control dilutions.

(9) Prepare a reference curve as directed below.

(II) AVAILABLE PLASMIN TEST (III) Active Plas-min Test (1) To preparethe plasma, add 1.0 ml. of the patients plasma to 0.1 ml. of agarose gelplate buffer.

(2) Proceed as directed in Steps 2-7 of I. Total Plasminogen Test.

(3) Replace the top cover of the plate and incubate at 37 '0. preferablyin a moist chamber, for at least 10 hours, or until clear reaction zonesappear for all plasma dilutions.

(4) Prepare a reference curve as directed below.

Preparation of a reference curve (1) After incubation of the clottedfibrin plate, measure the diameter of each reaction zone by placing theplate, with the cover removed, under the eyepiece of a HylandImmunoplate viewer. Align one side of the zone with the zero mark on thegrid and measure the diameter of the reaction zone to the nearest 0.1mm. Alternatively, a dissecting microscope with a stage micrometer oreyepiece reticle may be used.

(2) Using Z-cycle semilogarithmic graph paper, plot the reaction zonediameters of the three controls on the horizontal (arithmetic) scale andthe percent activity of the corresponding control on the vertical(logarithmic) scale. Draw a straight line of best fit.

(3) Determine the total plasminogen, availa'ble plasmin, or activeplasmin of the patient specimen by referring to the line formed by thecontrol. The level of fibrinolytic inhibitor may be calculated bysubtracting the available plasmin from the total plasminogenconcentration.

EXAMPLE 2 A clotted fibrin plate is prepared as in Example 1, exceptthat a predetermined amount of one unit per ml. of plasminogen is addedto the highly purified fibrinogen solution. The clotted fibrin plate isthen used for making a quantitative determination of plasminogenactivator. The procedure employed in carrying out this determination issimilar to that employed for Tests II and III in Example 1 except thatthe control sample of pooled human euglobulin contains additionally theplasminogen activator streptokinase of a known activity. The plasminogenactivator in the test specimen is determined by comparison with thecontrol sample as in Example 1.

Various other examples can be devised by the person skilled in the artWithout departing from the spirit and scope of the invention definedherein. All such further examples are within the scope of the inventionas defined in the appended claims.

What is claimed is:

1. A method for the quantitative determination of plasminogen in bloodplasma and fractions thereof comprising reacting said blood plasma orfraction thereof with clotted fibrinogen homogeneously suspended in astable gel medium at a concentration of from about 10 mg. percent toabout 2 gm. percent together with a predetermined amount of plasminogenfor a predetermined period of time to produce a clear radial difiusionzone of reaction product, measuring the diameter of said radialdiffusion zone and comparing with control samples of known plasminogenactivator activity, said blood plasma or fraction thereof being allowedto come into contact with said fibrinogen and plasminogen for reactiontherewith by diffusion from a well in the surface of said gel on aplate.

2. The method of claim 1 in which the gel medium contains from about1.0% to about 10% by weight of a gelling agent.

3. The method of claim 2 in which the gelling agent is agarose.

4. The method of claim 3 in which the gel medium contains about 2.5% byweight of agarose.

5. An agarose gel plate for the quantitative determination ofplasminogen activator in blood plasma and fractions thereof comprising aplate and an agarose gel medium containing from about 10 mg. percent toabout 2 gm. percent clotted fibrinogen together with a predeterminedamount of plasminogen homogeneously suspended in said gel medium.

References Cited UNITED STATES PATENTS 3,482,943 12/1969 Csizmas et al.-1035 R X OTHER REFERENCES Astrup et al., Archives of Biochemistry andBiophysics, vol. 40, pp. 346-351 (1952), QP 501.A77.

A. LOUIS MONACELL, Primary Examiner I. R. HOFFMAN, Assistant ExaminerUS. Cl. X.=R.

23-23O B; 195--103.5 R

